Aflatoxins, secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus, are natural toxic compounds that can cause various damages to human and livestock. So far, more than 20 varieties of aflatoxins have been discovered, including aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), AFG, and M1 (AFM1), and the like. Among these, AFB1 has the strongest toxicity. The toxicity of AFB1 is 10 times that of potassium cyanide and 68 times that of arsenic. Early in 1993, AFB1 was categorized as one of the most potent carcinogenic chemicals known to International Agency for Research on Cancer of the World Health Organization, i.e., class I carcinogen. China is more severely polluted by aflatoxin, which may even exist in various food and agricultural products, especially in corn, peanut, and their products. Therefore, it is significant to reinforce detection, especially quick detection, of aflatoxin and to keep track of health information on various food and agricultural products, so that food safety in China can be guaranteed.
The existing methods for detection of aflatoxin include thin-layer chromatography, instrumental analysis and immunology analysis. Thin-layer chromatography is a common method for detection of aflatoxin at early time, which does not require special instrument and can be conducted in an average laboratory. However, thin-layer chromatography requires large dosage of reagent and complicated procedure, is seriously interfered by other components, and has poor accuracy, thus can hardly quantify precisely. In addition, thin-layer chromatography causes severe contamination hazard to experimenters and the surrounding environment, and thus is unfit for on-site detection. Instrumental analysis mainly includes fluorescence spectrophotometry and high performance liquid chromatography, which have the advantages of high sensitivity and preferable accuracy. However, the above mentioned methods require high degree of purification of aflatoxin sample. Traditional sample pre-treatment technology, such as liquid-liquid extraction, solid phase extraction and solid phase microextraction, has complicated pre-treatment procedure and less specificity. In this case, the establishment of a fast and effective sample pre-treatment has become the primary and bottleneck problem of detection and analysis of aflatoxin. Immunoaffinity column is a new type efficient sample pre-treatment technology, which implements enrichment and purification of target substance in complex samples based on reversible bonding of specificities of antigen and antibody. Immunoaffinity column combined with liquid phase chromatographic analysis, fluorescence quick-detection device and ELISA method can be widely used in the detection of aflatoxin in agricultural products and food.
At present, aflatoxin nanobody immunoaffinity column is mainly prepared by coupling of traditional antibody (polyclonal antibody or monoclonal antibody) with sepharose gel and silica gel microparticles. Since activity of the traditional antibody degenerates fast during use, it is a technical problem that the immunoaffinity column available in the market can only be repeatedly used for limited number of times. Nanobody is heavy chain antibody naturally existing in camelidae animals. So far, there is not yet any report related to aflatoxin nanobody immunoabsorbent and immunoaffinity column.